1) Inactivation of all four alleles of Msx1 and Msx2 in neural crest cells leads to an unexpected formation of ectopic bone in the frontal bone region of mouse embryos. 2) This ectopic bone develops from neural crest cells that normally do not form bone and are located in the layer through which the frontal bone normally expands. 3) The formation of ectopic bone is associated with upregulation of Bmp signaling in this normally non-osteogenic neural crest cell layer. Inhibition of Bmp signaling prevents ectopic bone formation.

1. Inactivation of Msx1 and Msx2 in neural crest reveals an unexpected role in
suppressing heterotopic bone formation in the head
Paul G. Roybal, Nancy L. Wu, Jingjing Sun, Man-chun Ting, Christopher A. Schafer, Robert E. Maxson ⁎
Department of Biochemistry and Molecular Biology, Norris Cancer Hospital, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles,
CA 90089-9176, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received for publication 23 July 2009
Revised 6 April 2010
Accepted 7 April 2010
Available online 14 April 2010
Keywords:
Msx1
Msx2 neural crest
Heterotopic bone
Skull vault
Bmp
In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1
and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles
results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos
lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic
islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived
from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-
osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic
osteogenesis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by
implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These
results suggest that Msx genes have a dual role in calvarial development: They are required for the
differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress
an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of
this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of
pathological conditions in which calvarial bone development is compromised.
© 2010 Elsevier Inc. All rights reserved.
Introduction
The vertebrate skull vault consists of several component bones of
mixed lineage origin. In the mouse, these are the paired frontal and
parietal bones and a single interparietal bone. The frontal bones are
derived from neural crest, the parietal bones from mesoderm (Jiang
et al., 2002). The interparietal bone is a composite, its central portion
derived from neural crest and its lateral portions from mesoderm. The
patterned growth of the vertebrate skull is a complex and as yet
poorly understood process that involves an exquisitely controlled
series of migratory and proliferative events, as well as the specifica-
tion and differentiation of osteogenic and chondrogenic cells among
others (Chai and Maxson, 2006; Morriss-Kay and Wilkie, 2005).
During the first stages of skull vault development, precursor neural
crest and mesodermal cells migrate to positions on the lateral aspects
of the cerebral hemispheres. The frontal and parietal bone rudiments
become evident at E12.5 as condensations within these mesenchymal
cell populations. They express early osteoblast markers, and then
expand apically through the remainder of prenatal development and
the first several days of postnatal development, ultimately coming
into apposition with their paired counterparts at the midline (Ishii
et al., 2003; Jiang et al., 2002).
Two models have been put forward to explain the apical expansion
of the frontal and parietal bones (Lana-Elola et al., 2007; Ting et al.,
2009; Yoshida et al., 2008);. One posits proliferation and migration of
precursor cells located at base, the second, differentiation of
preexisting precursors. In favor of the first model, DiI injections into
the rudiment at E13.5, followed by exo utero culture of embryos
revealed that during apical expansion cells migrate from the base of
the rudiments to the leading edge of the growing bone. Cells within
the rudiment are also proliferating during this period of expansion.
Thus migration and proliferation are probably important morphoge-
netic forces contributing to the growth of the calvarial bones. Arguing
against the second model are results of diI injections into mesenchy-
mal cells located apical to the rudiment. Labeled cells do not have an
ostoegenic fate, but are located in a non-osteogenic layer flanking the
bone (Yoshida et al., 2008). We note, however, that when diI
injections are performed at late stages in the presumptive sagittal
suture, a small number of labeled cells are found in bone (Lana-Elola
et al., 2007). Thus at least some sutural cells are capable of
differentiating into osteoblasts. The emerging view of at least the
early stages of calvarial bone growth is that the bone rudiments grow
by end-addition of migratory cells through a layer of non-osteogenic
mesenchyme.
Developmental Biology 343 (2010) 28–39
⁎ Corresponding author.
E-mail address: maxson@usc.edu (R.E. Maxson).
0012-1606/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2010.04.007
Contents lists available at ScienceDirect
Developmental Biology
journal homepage: www.elsevier.com/developmentalbiology

2. Twist1, Msx1, and Msx2 have been shown to have major influences
on calvarial growth and patterning (Chai and Maxson, 2006). Twist1, a
basic helix–loop–helix transcription factor, is mutated in Saethre–
Chozen syndrome, characterized by craniosynostosis as well as other
craniofacial and limb defects (el Ghouzzi et al., 1997). Twist1 is
required for proper targeting of migratory osteogenic cells to the
leading edges of growing bones. This activity requires EphA4, which
functions in a layer of cells in which osteogenic precursors migrate,
flanking the prospective bone (Merrill et al., 2006; Ting et al., 2009).
Twist1, EphA4 and Twist1-EphA4 mutants exhibit inappropriate
migration of osteogenic cells into the coronal suture and consequent
differentiation of normally non osteogenic suture cells. The result is
synostosis of the frontal and parietal bones (Ting et al., 2009).
Msx genes function in the apical expansion of the frontal and
parietal bone rudiments (Han et al., 2007; Ishii et al., 2003). In Msx2
conventional mutants, the growth of the rudiments is retarded and
cells within the rudiments proliferate at a reduced rate (Ishii et al.,
2003). In combination Msx1/2 mutants, the frontal and parietal bones
do not form and many embryos exhibit exencephaly (Han et al.,
2007). The severity of this set of phenotypes precluded a detailed
analysis of the role of Msx genes in calvarial bone growth. This
limitation, together with the critical role of Msx genes in the apical
expansion of the rudiments, prompted us to undertake a more
detailed analysis of the activities of Msx1/2.
We produced floxed alleles of Msx1 and Msx2 (Fu et al., 2007). In
the present study, as part of an effort to understand more fully the
morphogenetic forces shaping calvarial bones, we inactivated Msx1
and Msx2 conditionally in neural crest. We show that Wnt1-Cre-
mediated inactivation of up to three Msx1/2 alleles results in a pro-
gressively larger frontal bone defect. Unexpectedly, in embryos
lacking all four Msx alleles, the large defect is largely filled with
bone, which is mispatterned and present in sutures. This bone is
derived from neural crest, not mesoderm, and, from diI cell marking
experiments, originates in the normally non-osteogenic layer of cells
through with the rudiment grows. Associated with the heterotopic
osteogenesis is an upregulation of Bmp signaling in this cell layer.
Inactivation of such signaling by implantation of noggin-containing
beads in calvarial explants prevents heterotopic osteogenesis. These
results, together with previous studies, suggest that Msx genes have a
dual role in calvarial development: They are required for the dif-
ferentiation and proliferation of osteogenic cells within rudiments
(Han et al., 2007; Ishii et al., 2003), and they are also required to
suppress an osteogenic program in a normally non-osteogenic cell
layer within which the rudiments grow.
Results
An unexpected regulative response in the frontal bones of Wnt1-Cre;
Msx1/2cko/cko
mutant embryos
We used Wnt1-Cre to produce a neural crest-specific knockout of
Msx1 and Msx2. We showed previously that Wnt1-Cre caused an
efficient knockout of each gene (Fu et al., 2007). Wnt1-Cre; Msx1cko/cko
;
Msx2cko/cko
embryos survived to the newborn stage and died shortly
thereafter, with cleft palate, which was fully penetrant (n=10). All
embryos examined (nN50) also exhibited a foreshortened mandible
and maxilla, making them easily recognizable.
We produced an allelic series of floxed Msx1 and Msx2 alleles
together with Wnt1-Cre, and examined the morphology of skulls at
the newborn stage (Fig. 1). The skulls exhibited a defect in the frontal
bone that became progressively larger as the number of functional
Msx alleles decreased. Analysis of different allelic combinations of
Msx1 and Msx2 revealed that the two genes were equivalent in their
effects on the frontal bone defect (Fig. 1; data not shown).
Since the calvarial bones do not develop in conventional Msx1/2
knockouts (Han et al., 2007), we expected that complete Wnt-Cre-
mediated inactivation of Msx1/2 would result in a more severe
foramen than in homozygous–heterozgyous combinations. Strikingly,
however, upon inactivation of the fourth Msx allele, a new phenotype
became evident: Alizarin-stained bone was present over much of the
area where we expected to see an unossified persistent foramen. This
phenotype was fully penetrant (10/10 skulls examined). Thus Msx1/2
mutant embryos “regulated” and partially repaired the frontal bone
defect. This regulative bone obliterated part of the frontal suture and
was irregular in shape, suggesting that it was not subject to normal
patterning mechanisms. In addition, the extent of apical growth of the
parietal bones was reduced in Msx1/2 homozygous mutant embryos.
This effect was non-autonomous since the parietal bones are derived
from mesoderm. Finally, double heterozygous Msx1/2 mutants had a
cleft in the posterior portion of the interparietal bone. In double
homozygous mutants, an additional defect was evident in the anterior
of the interparietal bone. Both defects occurred in the central portion
of the bone, which is derived from neural crest (Jiang et al., 2002).
We examined a developmental series of embryos to determine
when frontal bone regulation was first detectible. We stained
embryos in whole mount for the early osteoblast marker, alkaline
phosphatase (ALP) (Fig. 2). In control embryos at E12.5, the frontal
bone rudiment was evident as a crescent of ALP-stained cells in the
supraorbital ridge. Immediately posterior to the rudiment is an ALP-
free area corresponding to the presumptive coronal suture; posterior
to the suture is the parietal bone rudiment. In Msx1/2cko/cko
mutants,
no staining was apparent in the area of the frontal bone rudiment nor
was there staining on the apical portion of the head in the area where
the regulative bone would later form.
At E13.5, as revealed by ALP staining, the frontal and parietal bone
rudiments of control embryos were significantly larger than at E12.5
(Fig. 2). No ALP activity was present apical to the rudiments. In
contrast, in Msx1/2cko/cko
; Wnt1-Cre mutants, the ALP-positive area
was irregular in shape, and extended apically to a much greater
degree than controls. By E14.5, there was extensive regulative bone in
the Msx1/2 mutants. The parietal bone was substantially smaller than
in the wild type embryo. Thus the inactivation of Msx1/2 in the frontal
bone territory influenced the growth of the parietal bone during
embryogenesis.
Regulative frontal bone in Wnt1-Cre; Msx1/2 mutants is derived from a
population of neural crest whose normal fate is non-osteogenic
To understand the processes that gave rise to the regulative bone in
Msx1/2 mutants, we sought to determine its tissue of origin. We
considered two possibilities. The first was that the bone arose from
mesoderm-derived cells that normally form the parietal bone. The
close proximity of the regulative bone to the parietal bone made this an
attractive possibility. Also consistent with this possibility is the finding
that regulative bone does not occur in the frontal bone territory of
conventional Msx1/2 knockouts (Han et al., 2007), suggesting that its
development may depend on Msx gene function in a non-neural crest
cell type.
We crossed the R26R marker allele into Wnt1-Cre;Msx1/2 mutants
and examined the distribution of lacZ positive cells in double
homozygous conditional mutant and control embryos at E13.5
(Fig. 3). In control embryos, a lacZ positive layer of loose mesenchyme
was evident between the epidermis and the meninges. This layer is
composed of neural crest cells that migrate apically between E9.5 and
E10.5, prior to the growth of the frontal bone rudiment (Ishii et al.,
2003); we refer to these cells as “early migrating” neural crest cells.
This layer is continuous with the ALP-positive frontal bone rudiment.
At E12.5, Msx1 and Msx2 are coexpressed in this layer (Fig. 4) (Ishii
et al., 2003; unpublished observations). At E13.5, Msx1 is expressed
predominantly in the meninges and Msx2 in the overlying mesen-
chyme (Fig. 4).
29P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

3. In wild type embryos, the apical portion of the early migrating
neural crest layer did not stain appreciably for ALP (Fig. 3B). In
contrast, in mutant embryos, the regulative prospective bone was
detectible as a patchy ALP stained layer apical to the frontal bone
rudiment (Fig. 3D). Cells of this layer appeared more tightly
condensed than their wild type counterparts. Staining for lacZ
revealed that these ALP-positive cells were entirely or almost entirely
of neural crest origin (Fig. 3E–H). Thus, although the regulative
response may require Msx gene function in a non-neural crest cell
type, it does not entail recruitment of mesoderm-derived cells to the
frontal bone defect.
BrdU incorporation experiments revealed that the proportion of
BrdU positive cells in the apical portion of the early migrating neural
crest was not significantly different from that of their counterparts in
control embryos at E12.5 (Supplementary Fig. 1) or E13.5 (data not
shown), although there was a 17±5% increase in total cell number.
Cells of the early migrating mesenchyme do not undergo apoptosis at
an appreciable rate in wild type embryos (Ishii et al., 2003; our
unpublished observations); therefore the increase in total cell number
is likely due to an earlier event in neural crest development.
We next addressed the source of the ALP-positive, neural crest-
derived cells that form the regulative bone of Msx1/2 mutants (Fig. 5).
The simplest possibility was that the cells originated from the early
migrating neural crest. It was also possible that they were the result of
an aberrant recruitment of the neural crest-derived osteogenic
precursor cells that normally migrate from the supraorbital ridge to
the leading edge of the growing frontal bone (Yoshida et al., 2008;
Ting et al., 2009). To test these possibilities, we carried out a series of
diI cell marking experiments.
We labeled cells of the early migrating neural crest layer and asked
whether they became incorporated into heterotopic bone (Fig. 5). DiI
was injected into mutant or control embryonic heads at E13.5, during
the early stages of the formation of heterotopic bone. The dye was
placed apically, beyond the dorsal margin of bone at this stage (see
Fig. 2). Embryos were allowed to develop exo utero and then
examined for the distribution of the dye (Fig. 5). In mutant embryos
at E16.5, the dye was located almost exclusively in ALP-expressing
cells of heterotopic bone. In control embryos, in contrast, the dye
became localized in a cell layer flanking the bone (Fig. 5; Table 1).
These results suggest that in Wnt1-Cre; Msx1/2cko/cko
mutants at
E13.5, cells of the early migrating layer of neural crest are allocated to
form heterotopic bone.
To determine whether neural crest-specific inactivation of Msx1/2
caused changes in the apical migration of osteogenic precursor cells
from the area of the frontal bone rudiment, we carried out DiI labeling
of the frontal bone rudiment at E13.5 and assessed the distribution of
dye at E16.5 (Fig. 6). This migration has been documented in detail
(Yoshida et al., 2008; Ting et al., 2009). We used the same diI injection
protocol as in our previous work (Ting et al., 2009) to assess migration
in Msx1/2 mutants. In control embryos, labeled cells were found in
both the ectocranial layer in which the cells migrate, and in the
developing bone, which is their ultimate fate. We did not detect a
significant change in the number of labeled migratory cells in mutant
embryos. These results suggest that the apical migration of osteogenic
Fig. 1. Dual functions of Msx1/2 in promoting frontal bone development and suppressing heterotopic bone formation in early migrating cranial mesenchyme. Skulls of Wnt1-Cre;
Msx1cko/cko
; Msx2cko/cko
mutants at the newborn stage were stained with Alizarin Red to reveal bone. A, wild type; B, Msx1cko/+
;Msx2cko/+
; C, Msx1cko/+
;Msx2cko/cko
; D, Msx1cko/cko
;
Msx2cko/+
; E, Msx1cko/cko
; Msx2cko/cko
. At least five skulls of each genotype were examined. We show representative images. Note increasing size of frontal foramen with decreasing
Msx gene dosage up to homozygote–heterozygote combination (arrows). Note unpatterned, heterotopic bone in area of posterior frontal bone in E (bracket).
30 P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

4. precursor cells from the supraorbital ridge does take place in Msx1/2
mutants. However, our finding that placement of dye in the early
migrating mesenchyme in the apical portion of the head at E13.5
results in virtually 100% of the label in bone suggests that the principal
source of the heterotopic bone is the early migrating mesenchyme,
not the migratory cells of the frontal bone rudiment.
Timing of action of Msx genes
DiI labeling suggested that in Wnt1-Cre/Msx1/2 mutants, the early
migrating neural crest cells are mis-allocated to form bone as early as
E13.5. Thus Msx genes must be required for the proper allocation
of this cell layer at or before E13.5. To determine when Msx genes
Fig. 2. Prospective heterotopic bone is detectible at E13.5. Embryonic heads were stained in whole mount (A–F) or in cross sections (G–L) for alkaline phosphatase (ALP activity at
E12.5 (A, B, G, H) and E13.5 (C, D, I–L) and E14.5 (E, F). At least three mutant and three control embryos were examined at each stage. Shown are representative images. Dotted lines
in whole mount figures indicate planes of section in Figs G–L. Note that in the Wnt1-Cre; Msx1/2 cko/cko
mutant at E12.5, the frontal bone rudiment is not detectible (arrows, B, H). At
E13.5, an area of ALP stain is evident apical to the eye, extending approximately 2/3 of the distance to the dorsum of the head (D, arrows). Sections show an ALP positive layer in the
apical region of the head (K, L, arrows). Fb, frontal bone; pb, parietal bone; ip, interparietal bone, cs, coronal suture.
31P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

5. are required to suppress heterotopic bone formation, we used a
Tamoxifen-inducible Cre mouse line (Hayashi and McMahon, 2002).
We produced mice carrying the Cagg-ER transgene together with
floxed alleles of Msx1 and Msx2. We used a dosage of tamoxifen that
we determined to be sufficient to cause efficient recombination of the
R26R marker allele within 24 h of injection at each of the time points
(Hayashi and McMahon, 2002; our unpublished observations).
We injected tamoxifen into pregnant females at 24-h intervals
from 9.5 days pc to 12.5 days pc and examined newborns for het-
erotopic bone in the area that the frontal bone would normally
occupy. We found that Cagg-ER-Cre; Msx1cko/cko
; Msx2cko/cko
embryos
injected at E9.5 had small areas of heterotopic bone; embryos injected
at E10.5 had substantially larger areas (Fig. 7). Embryos injected at
E11.5 or E12.5 did not have heterotopic bone. Given that complete
Cre-mediated recombination occurs approximately 24 h after injec-
tion of tamoxifen (Hayashi and McMahon, 2002), the interval during
which Msx1/2 function to suppress heterotopic bone formation is
between E10.5 and E11.5. This is the interval during which the early
migrating neural crest migrates apically (Ishii et al., 2003).
Bmp signaling is necessary for heterotopic ossification in Msx1/2 neural
crest-specific mutants
The well-documented role of Bmp signaling in osteogenesis and
the known relationship between Msx genes and the Bmp pathway
(Brugger et al., 2004; Chai and Maxson, 2006; Maxson and Ishii, 2008)
prompted us to examine the status of Bmp signaling during
heterotopic bone formation in Wnt1-Cre/Msx1/2 conditional mutants
(Fig. 8). We assessed the expression of Bmp2 and Bmp4, both known
to be expressed in calvarial tissues (Kim et al., 1998). In addition we
examined the distribution of P-Smad1/5/8 as an indicator of the net
activity of canonical Bmp signaling. In wild type embryos at E12.5,
Bmp2 was expressed in the meningeal layer and at a low level in the
overlying mesenchyme. Bmp4 was also expressed in the meningeal
layer and at a much higher level in the overlying mesenchyme. In
Wnt1-Cre; Msx1/2 cko/cko
mutants Bmp2 expression in the early mi-
grating mesenchyme increased and Bmp4 expression decreased. A
similar result was evident at E13.5. Immunostaining for P-Smad1/5/
8 at E13.5 showed elevated levels in the early-migrating mesenchyme
layer, indicating that the net change in Bmp signaling was positive.
The upregulation of Bmp signaling in Msx mutants in association with
the development of heterotopic bone suggests that the Bmp pathway
has a role in this process.
To test directly whether the increase in Bmp signaling is necessary
for heterotopic bone development in Msx mutants, we carried out a
series of bead implantation experiments in cultured calvarial explants
(Fig. 9). We implanted beads containing either the Bmp inhibitor,
noggin, or BSA, in explants of Msx1/2 mutant and control heads taken
at E13.5. Beads were placed in several locations along the apical-basal
axis in order to ensure that at least one bead would be located in the
area of heterotopic bone (Fig. 9A). Explants were cultured for 48 h,
then sectioned and stained for ALP activity. Results representative of
three repetitions of this experiment are shown.
In mutant heads that did not receive beads, heterotopic ALP stain
was apparent in the apical region, in the mesenchymal layer beneath
the surface ectoderm (Fig. 9E). No such stain was evident in control
heads (Fig. 9B). Apical placement of noggin beads in mutants resulted
in reduced ALP staining in the mesenchyme surrounding the bead
(Fig. 9G). BSA beads did not produce this effect (Fig. 9F). These data
suggest that Bmp signaling is required for the development of
heterotopic calvarial bone in Wnt1-Cre; Msx1/2 mutants.
Discussion
Here we demonstrate that Msx1 and Msx2, shown previously to be
required for calvarial bone development (Han et al., 2007; Ishii et al.,
2003), also suppress bone formation in a head tissue that is normally
non-osteogenic. Whereas inactivation of up to three Msx1/2 alleles by
means of Wnt1-Cre results in a progressive increase in size of a defect
in the frontal bone, inactivation of the final Msx1/2 allele results in the
Fig. 3. Neural crest origin of heterotopic bone. To visualize neural crest-derived cells, we produced mice carrying the R26R marker allele along with Wnt1-Cre; Msx1cko/cko
; Msx2cko/cko
.
Embryos were taken at E13.5, and heads were sectioned in the coronal plane. Adjacent sections were stained either for lacZ (A, C, E, G) or ALP (B, D, F, H). Boxed areas in A–D correspond
to areas of heterotopic ALP activity (see Fig. 2). Arrows in G and H point to heterotopic prospective bone. Images shown are representative of three control and three mutant embryos
examined. Note that the ALP-positive cells are in a lacZ positive cell layer and are therefore derived from neural crest. fb, frontal bone; b, brain; em, early-migrating neural crest; d,
dura; e, epidermis.
32 P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

6. filling in of the defect with disorganized bone. Thus the suppression of
such regulative bone formation requires a single allele of Msx1 or
Msx2. This bone is derived from neural crest, not mesoderm, and
develops largely if not entirely from the early migrating neural crest, a
population of neural crest cells that migrates prior to those that
normally compose the frontal bone. Finally, we demonstrate that
Wnt1-Cre-Msx1/2 mutant embryos exhibit dysregulation of Bmp2 and
Bmp4 expression, resulting in increased P-Smad1/5/8 activity in the
early migrating neural crest. Moreover, we show that noggin-soaked
beads can abrogate the formation of heterotopic bone in explanted
heads of mutant embryos. Thus Bmp signaling is required for het-
erotopic bone development.
In conventional Msx1/2 homozygous knockout embryos, neural
crest cells migrate to the supraorbital ridge (site of the calvarial
rudiments) in near-normal numbers as judged by Wnt1-Cre/R26R
lineage tracing (Han et al., 2007; Ishii et al., 2003). Subsequently
neural crest cells of the frontal bone rudiments exhibit impaired
proliferation and differentiation, as well as an increase in apoptosis
(Han et al., 2007). The end result is that the frontal bones do not
develop. Given this profound deficiency, it is perhaps surprising that
the growth of the frontal bones is not more compromised in Wnt1-
Cre; Msx1/2cko/cko
mutants in which rudiments do develop after a
delay in the initial appearance of ALP-positive cells. Wnt1-Cre
produces an efficient inactivation of the Msx1 and Msx2 alleles used
in this study (Fu et al., 2007); therefore an incomplete knockout is not
a likely explanation. Instead, the discrepancy suggests that non-neural
crest tissues are likely to have a role in the development of the neural
crest-derived portion of the skull vault. A candidate tissue is a
population of mesoderm that flanks the frontal bone rudiment
ectocranially. Our results also provide evidence of an influence of
neural crest on mesoderm: the growth of the parietal bone rudiment
(mesoderm-derived) is impaired in Wnt1-Cre; Msx1/2cko/cko
mutants
(Fig. 2). It is noteworthy that the dura underlying the anterior portion
of the parietal bone is derived from neural crest (Jiang et al., 2000).
The dura is known to influence cranial suture development (Opper-
man et al., 1995) and could thus be part of a mechanism that controls
parietal bone growth.
Given the requirement for Msx1 and Msx2 for the growth of the
calvarial bones (Chai and Maxson, 2006; Maxson and Ishii, 2008), the
bone-suppressive activity in the Wnt1-Cre-mediated Msx1/2 knock-
out was a surprise. The appearance of the heterotopic bone in the area
normally occupied by the posterior frontal bone led us to predict that
Fig. 4. Expression of Msx1/2 in early migrating cranial mesenchyme. Coronal sections of embryos at E12.5 and E13.5 were stained for ALP activity (A, G) and incubated with Msx1
(B, D, H, J) and Msx2 (C, E, I, K) probes simultaneously. Hybridization signals were visualized by immunofluorescence. Msx1 is in red, Msx2 in green. D, E, J and K show boxed areas in
B, C, H and I. F and L are merges of images in D, E, J and K respectively. Note partial overlap of Msx1 and Msx2 signals in the neural crest-derived mesenchyme layer at E12.5 (yellow
color, arrowhead, F). At E13.5, Msx1 is expressed in the meninges, internal to Msx2 (L, arrowheads). Msx2 is expressed in the mesenchymal layer (K).
33P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

7. mesodermal cells normally allocated to the parietal bone were
migrating into the defect and forming bone. However, Wnt1-Cre
mapping showed that the bone is entirely of neural crest origin. Thus
the heterotopic bone appears to depend on the autonomous function
of Msx genes in the neural crest. Since the heterotopic bone does not
occur in conventional Msx1/2 knockouts (Han et al., 2007), its
development may also depend on Msx gene function in a non-neural
crest cell type.
From what subpopulation of neural crest does the heterotopic
bone arise? Clues came from recent work on the mechanism of
growth of the skull vault. The frontal and parietal bones grow by
end-addition of migratory precursors (Ting et al., 2009; Yoshida et
al., 2008). The bone rudiments grow through a pre-existing layer of
mesenchyme. In the case of the frontal bone, cells of this layer are
derived from neural crest, and are designated “early migrating
neural crest.” DiI labeling of cells in this mesenchyme layer showed
that they are not allocated to become bone (Yoshida et al., 2008).
Instead, after the calvarial bone rudiments have elongated to the
apex of the head, DiI-labeled cells are found in a layer that flanks
the bone ectocranially. Intriguingly, DiI labeling of Wnt1-Cre; Msx1/
2 mutants showed that cells of this layer are incorporated into bone.
Thus the early migrating neural crest forms heterotopic bone. To
what extent migratory osteogenic precursor cells originating in the
supraorbital ridge contribute to heterotopic bone is not clear. DiI
labeling experiments suggest that the rudiments produce migratory
cells normally. However, the fact that virtually 100% of the labeled
cells in the early migrating mesenchyme are ultimately found in
bone suggests the early migrating mesenchyme is the principal
source of the heterotopic bone. These results imply a change in the
fate map of the early migrating mesenchyme in Wnt1-Cre; Msx1/2
mutants, although we stress that we have not shown directly that
individual cells of the early migrating layer convert to an osteogenic
fate. BrdU labeling experiments showed no change in the
proliferation index of the early migrating mesenchyme of Wnt1-
Cre; Msx1/2 mutants, but did reveal an overall increase in cell
number of approximately 20% at E12.5. It is possible that this
increase promotes condensation of the early migrating cells and
thus leads to osteogenic differentiation.
Our data strongly suggest that the action of the Bmp pathway is at
least part of the cause of the ectopic bone. Analysis of Bmp2 and Bmp4
expression showed a loss of Bmp4 expression and an upregulation of
Bmp2 expression in the early migrating neural crest at E12.5, prior to
the detection of ectopic ALP. No change was detected in the ex-
pression of noggin (data not shown). Thus the observed increase in
the number of P-Smad 1/5/8 positive cells at E13.5 is likely a result of
the changes in Bmp2 and Bmp4 expression. It is interesting that Bmp2
and Bmp4 appear to have subtly different spatial patterns of
expression: Bmp4 is expressed in the meningeal layer as well as in
the overlying mesenchyme; Bmp2 is expressed predominantly in the
Fig. 5. Inactivation of Msx1/2 in neural crest causes a change in the fate of early migrating cranial mesenchyme. We injected DiI into heads of E13.5 control and Msx1/2 cko/cko
; Wnt1-
Cre and control embryos and assessed the distribution of dye after exo utero development until E16.5. Dye was placed near the apex, in the area in which the frontal bone will
develop in control embryos and heterotopic bone will develop in Msx1/2cko/cko
; Wnt1-Cre mutants. The placement of dye is shown in a representative embryo in A, and schematically
in B and C. Embryos were allowed to develop to E16.5, and were then sectioned in the coronal plane (D, see also Fig. 2) and photographed (E–J). E and H are epifluourescence images,
F, I, brightfield images, G, J, merged images. In control embryos, dye was distributed in a layer of cells flanking the prospective bone. Few if any labeled cells were found in the
prospective bone (arrowheads). In mutant embryos, dye was located largely in the developing bone. We obtained substantially similar results in several repetitions of this
experiment (Table 1).
Table 1
Location of labeled cells following diI injection into calvarial rudiments or apical
mesenchyme.
Experiment Genotype Site of
injectiona
Location of
labeled cellsb
1–10 Controlc
r e, b
11 Msx1cko/cko
; Msx2cko/cko
r e, b
11 Controlc
r e, b
11 Control r e, b
12 Msx1cko/cko
; Msx2cko/cko
r e, b
12 Msx1cko/cko
; Msx2cko/cko
r e, b
12 Controlc
r e, b
12 Controlc
r e, b
13 Msx1cko/cko
; Msx2cko/cko
r e, b
13 Controlc
r e, b
13 Controlc
r e, b
14 Controlc
a e
14 Controlc
a e
14 Msx1cko/cko
; Msx2cko/cko
a b
15 Msx1cko/cko
; Msx2cko/cko
a b
15 Control a e
16 Control a e
16 Msx1cko/cko
; Msx2cko/cko
a e
a
diI injection is described in Fig. 5; r: frontal bone rudiment, a, apical mesenchyme.
b
Embryos were analyzed after 48 h of exo utero development; e, ectocranial
mesenchyme, b, prospective frontal bone.
c
Genotypes of control embryos were either wild type, or Wnt1-Cre; Msx1/2 double
heterozygotes, which had no phenotype.
34 P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

8. mesenchyme layer. Thus a critical change in the Msx1/2 neural crest-
specific knockout may be the loss of Bmp signaling in the meningeal
layer.
A definitive link between Bmp signaling and heterotopic bone
came from experiments in which we implanted noggin-soaked beads
under the skin of explanted Msx1/2 mutant and control heads. This
approach showed that noggin inhibited the appearance of ALP-
positive cells in the early migrating neural crest layer. Thus Bmp
signaling, downstream of Msx1/2, is required for heterotopic bone
formation in the head. It is perhaps surprising that previous work on
the conventional Msx1/2 knockout did not demonstrate a change in
Bmp2 or Bmp4 expression at E12.5 in mutant embryos (Han et al.,
2007). We do not know the reason for this discrepancy, although it is
reasonable to expect that in the germline knockout, compensatory
mechanisms operating over all of embryogenesis might damp effects
of Msx1/2 inactivation on Bmp gene expression in neural crest-
derived tissues.
Heterotopic bone formation driven by Bmp signaling is also
observed in fibrodysplasia ossificans progressiva, a disorder charac-
terized by progressive heterotopic ossification in which skeletal
muscles and connective tissues are transformed into bone (Billings et
al., 2008). This disorder is associated with a specific mutation in
ACVR1, which encodes a bone morphogenetic protein type I receptor.
The ectopic skeleton is derived largely from cells of vascular origin
(Billings et al., 2008).Whether Msx genes have a role in fibrodysplasia
ossificans progressiva has not been reported.
The ectopic bone in Msx1/2 mutants bears some resemblance to
Wormian bones, defined as cranial bones that have no relationship to
the normal ossification centers driving skull growth (Parker, 1905;
Sanchez-Lara et al., 2007). Wormian bones often occur in sutures;
however, they can be more extensive, as in osteogenesis imperfecta in
which the calvarial bones are sometimes largely replaced by a mosaic
of Wormian bones (Baljet, 2002). Wormian bones can also be the
result of an unusually large head, which increases the time required
for the frontal and parietal bones to grow from rudiments in the
supraorbital ridge to the skull apex (Sanchez-Lara et al., 2007.
Wormian bones are thus, in the most general sense, a regulative
response to a deficiency in calvarial bone growth. We suggest that
Msx-dependent heterotopic bone can viewed similarly. Whether such
heterotopic bone is truly Wormian bone is not clear: The tissue of
origin of Wormian bone has not been investigated. However, it is
suggestive that the two are not only similar morphologically but also
likely result from a regulative response to compromised calvarial bone
growth.
Finally, we are intrigued by the potential evolutionary implications
of the Msx1/2-dependent program we have uncovered. Our results,
together with recent DiI cell marking experiments; (Ting et al., 2009;
Yoshida et al., 2008), document two distinct osteogenic programs in
skull vault development. One consists of the patterned growth of the
calvarial rudiments by end addition of migratory osteogenic precursor
cells. The other consists of the differentiation of the early migrating
mesenchyme along an osteogenic pathway. The former appears to be
the primary mechanism by which the frontal and parietal bones grow.
The latter mechanism, we suggest, may be an evolutionary remnant of
a program present in basal vertebrates with a mode of calvarial bone
growth distinct from that of mammals. Skulls of fish typically have
many loosely connected elements; those of mammals are more tightly
connected and have fewer elements (de Beer, 1985; Romer, 1997).
These changes in skull structure must be a result of changes in the
developmental program underlying skull growth. One scenario is that
the osteogenic program in the early migrating mesenchyme
was the primitive condition, accounting for the multiplicity of loose-
ly patterned skull bones in early vertebrates. The migratory
program then arose secondarily. The loss of bones would then be a
result of suppression of the primitive program—e.g., by acquisition of
Msx1/2-dependent repression of osteogenesis in specific regions—
accompanied by an expansion of the growth by end addition. Analysis
of cranial development in extant agnathans may shed light on this
scenario.
Materials and methods
Mouse mutants and genotyping
Mutant lines were maintained in a C57Bl/6 mixed background.
The R26R (Soriano, 1999), Wnt1-Cre (Danielian et al., 1998) and Msx1
and 2 Floxed (Fu et al., 2007) alleles have been described. We
genotyped Msx1 and 2 Floxed, R26R, and Wnt1-Cre alleles by PCR as
described (Fu et al., 2007; Jiang et al., 2002).
Histology, immunostaining and in situ hybridization
Heads of embryos were embedded in OCT medium (Histoprep,
Fisher Scientific) before sectioning. Frozen sections were cut at 10 μm.
Fig. 6. Osteogenic precursor cells can migrate apically from the supraorbital ridge in Msx1/2 cko/cko
; Wnt1-Cre mutant embryos. To assess the apical migration of osteogenic precursor
cells, we injected DiI in the supraorbital ridge of control and mutant embryos at E13.5 as shown in A. Embryos were allowed to develop exo utero until E16.5, and were then sectioned
in the indicated plane (B) and photographed. Note labeled precursor cells in the ectocranial layer (e, arrowheads) as previously described (Ting et al., 2009; Yoshida et al., 2008).
These cells add to the leading edge of the growing bone (b) in both control and mutant embryos, suggesting that this morphogenetic mechanism is functional in Msx1/2cko/cko
; Wnt1-
Cre mutants. We obtained substantially similar results in several repetitions of this experiment (Table 1).
35P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

9. Analysis of β-galactosidase activity of Wnt1-Cre/R26R reporter gene
expression was carried out as described (Ishii et al., 2003).
Immunostaining of frozen sections was largely carried out as
previously reported (Ishii et al., 2003). Immunohistochemistry was
performed using rabbit anti-P-Smad1/5/8 (pSmad1/5/8) (Cell
Signaling) or rabbit anti-phosphorylated Histone3 (pH3) (Cell
Signaling) diluted in 1%BSA/PBS and incubated overnight at 4 °C.
Detection of anti-p-Smad1/5/8 or anit-pH3 was performed by
incubating rhodamine-labeled goat anti-rabbit IgG (1:100 for anti-
pSmad1/5/8) or (1:50 for anti-pH3) for1 h at room temperature
followed by DAPI counterstaining and examination by epifluores-
cence microscopy. Non-radioactive section in situ hybridization using
the tyramide signal amplification (TSA) method was performed as
described (Adams, 1992; Paratore et al., 1999; Yang et al., 1999).
Briefly, to analyze mRNA expression by TSA, DIG-labeled or F-labeled
riboprobes were allowed to hybridize with the section and were
detected with anti-DIG or anti-FL antibodies conjugated to horserad-
ish peroxidase (POD). Indirect TSA fluorescence system (TSA-biotin/
avidin-FITC) was used to detect the POD-conjugated antibody (Perkin
Elmer). RNA probes were generated as described (Yang et al., 1999):
the Bmp4 probe was DIG-labeled and Bmp2 FL-labeled (courtesy of Dr.
Malcolm Snead).
Whole-mount skull Alizarin Red S and ALP staining
Skulls from P0-day-old postnatal mice were stained for bone with
2% Alizarin Red S in 1% KOH for 1 to 2 days. The specimens were then
cleared and stored in 100% glycerol. Whole-mount staining for alkaline
phosphatase was carried out as described (Ishii et al., 2003). E12.5-
E14.5 embryonic heads were fixed in 4% paraformaldehyde in PBS, and
were bisected midsagitally after fixation. Presumptive calvarial bones
were stained with NBT and BCIP (Roche).
Exo utero DiI labeling
Details of the exo utero manipulation have been described
(Muneoka et al., 1986; Serbedzija et al., 1992). Briefly, E13.5 embryos
with embryonic membranes were carefully exposed by incising the
uterine wall. Two embryos from each side of the uterine horns were
designated as the experimental group, and all others were removed.
DiI (Molecular Probes, 1:10 dilution from 0.5% stock solution) was
injected into the area of the frontal bone rudiments under a dissecting
microscope with a microelectrode (tip diameter, 20 μm) attached to a
mouth pipette (Yoshida, 2005). After injection, the embryos were
returned to the peritoneal cavity of dams and allowed to continue
development exo utero. After 2–3 days of additional development, the
embryos were removed and examined by epifluorescence microsco-
py. The survival rate of the embryos after DiI injection was greater
than 70%.
BrdU labeling
Pregnant mice injected intraperitoneally with BrdU (200 µg/gram
of body weight) were sacrificed 2 h after the injection. Embryonic day
12.5 (E12.5) embryo heads were fixed in 4% paraformaldehyde and
were embedded in OCT medium (Histoprep, Fisher Scientific).
Immunodetection of BrdU was performed according to the manufac-
turer's instructions (Zymed). BrdU-positive cells in the area apical to
Fig. 7. Timing of Msx1/2 gene action in heterotopic bone formation. We used a Tamoxifen-inducible (Cagg-Er-Cre) to inactivate Msx1/2 at different times during development.
Tamoxifen was injected IP into pregnant females at the indicated times post coitum. Pups were taken at the newborn stage and bones of the skull vault visualized by staining with
Alizarin Red S. Note that Tamoxifen injected at E10.5 caused heterotopic bone (arrows). Tamoxifen injected at E11.5 and E12.5 resulted in retarded growth of the frontal and parietal
bones, but not heterotopic bone. Thus Msx1/2 are required between E10.5 and E11.5 to suppress heterotopic bone formation. At least three skulls at each time point were examined,
with substantially similar results.
36 P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

10. the frontal bone rudiment were counted in three mutant and three
wildtype littermate embryos. At least three sections were counted for
each embryo examined.
Organ culture and bead implantation
Organ culture of embryonic heads was carried out essentially as
described by Mohammad et al. (2008) and Hu and Helms (2001).
Briefly, control and Wnt1-Cre; Msx1/2cko/cko embryos were har-
vested at E13.5. Embryos were decapitated and the tongue and lower
jaw were removed. Affigel agarose beads were washed with sterile
PBS and allowed to dry before incubating them at 37 °C for 30 min in
either 0.1% BSA or 100 ng/ml noggin (R&D). Beads were implanted
under the skin, and skulls were placed on stainless steel grids which
were then inserted into the well of an organ culture dish (Falcon)
filled with BGJb medium supplemented with 0.1% BSA and 1U/ml
penicillin and streptomycin. The skulls were incubated at 37 °C for
48 h.
Acknowledgments
We dedicate this paper to the memory of Dr. Paul G. Roybal, a
young scientist with enormous talent, heart, and courage. Paul battled
brain cancer throughout earning his PhD and passed away on July 25,
2009, shortly after obtaining his degree. Paul's family wishes to thank
Dr. Jing-jing Sun, Dr. Nancy Wu, Dr. Mandy Ting, Dr. Mamoru Ishii, and
particularly Dr. Robert Maxson and Dr. Deborah Johnson for
supporting and mentoring Paul as he finished this work. The authors
Fig. 8. Dysregulated Bmp signaling is correlated with heterotopic bone in Wnt1-Cre; Msx1/2 mutants. We examined the expression of ALP (A, D, G, J), Bmp4 (B, E, H, K), Bmp2 (C, F, I, L),
and P-Smad1/5/8 (N) in the apical cranial mesenchyme at E12.5 and E13.5. ALP was detected by histochemistry, Bmp2 and Bmp4 by in situ hybridization, P-smad1/5/8 by
immunostaining. Boxed areas are shown in higher magnification in the image to the right. Note reduced expression of Bmp2 and increased expression of Bmp4 in the mesenchymal
(ALP-expressing) layer in Wnt1-Cre; Msx1/2cko/cko
mutants. Also note increase in P-Smad1.5.8-positive nuclei (N).
37P.G. Roybal et al. / Developmental Biology 343 (2010) 28–39

11. also thank Dr. Randall Widelitz, Damon De La Cruz, and Cathleen Chiu
for help with fluorescence microscopy. This work was supported by
NIH Grants DE19650 and DE16320 from the NIDCR to REM. PR was
supported by NIH Training Grant 5T32 GM067587.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ydbio.2010.04.007.
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